CN114127110A - anti-CGRP antibodies and uses thereof - Google Patents

Abstract

Disclosed is a novel antibody, particularly an antibody or antigen-binding fragment thereof that binds CGRP antigen, as well as methods of making, compositions and uses thereof. The antibody provided by the invention has stronger binding capacity with the CGRP antigen, low immunogenicity and long in-vivo half-life, and can be used for treating and/or preventing CGRP related diseases such as headache; it can also be used for immunological detection of CGRP antigen.

Description

anti-CGRP antibodies and uses thereof Technical Field

The invention relates to the technical field of biomedicine or biopharmaceutical, in particular to an anti-CGRP antibody or an antigen-binding fragment thereof, and a preparation method, a composition and application thereof.

Background

Calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide secreted by nerve cells of the central and peripheral nervous systems. In humans, CGRP exists in two forms: CGRP α and CGRP β, which differ by only 3 amino acids, and are derived from different genes (Doods, h., curr. op. invest. drugs,2(9):1261-78 (2001)). CGRP is a neurotransmitter released by a variety of tissues (e.g., trigeminal nerves) that, when activated, releases neuropeptides in the meninges, thereby mediating neurogenic inflammation characterized by vasodilation, vascular leakage, and mast cell degeneration (Durham, p.l., New eng.j.med.,350(11):1073-75 (2004)). The biological action of CGRP is mediated by CGRP receptors consisting of seven transmembrane component binding receptor associated proteins (RAMP) that further require the activity of a Receptor Component Protein (RCP) that is essential for efficient coupling to adenylate cyclase via G proteins and cAMP production. CGRP is a potent vasodilator associated with the pathology of other vasomotor symptoms, such as all forms of vascular headache, including migraine (with or without aura) and cluster headache. CGRP levels in the external jugular vein were elevated during migraine headache in patients (Goadsby et al, Ann. neurol.28:183-7 (1990)). Intravenous administration of human α -CGRP induces headaches and migraines in patients with migraine headaches without aura, suggesting that CGRP has a causal role in migraine (Lassen et al, Cephalalgia 22:54-61, (2002)).

Currently, three antibody products are approved for the CGRP target. Fremanzumab under the trade name

Developed by Teva, approved by the U.S. Food and Drug Administration (FDA) for marketing, is a humanized monoclonal antibody targeting calcitonin gene-related peptide (CGRP) and is approved for the treatment of adult migraine (Uris, Ivan et al. pain and therapy vol.9,1(2020): 195-). 215.). Eptinezumab, trade name

Is a humanized monoclonal antibody targeting calcitonin gene-related peptide (CGRP), blocks the binding of CGRP to its receptor and is approved for the prophylactic treatment of adult migraine (Dhillon S.2020; 80(7):733 739.). Galcanezumab, trade name

Developed by the Gift company, approved by the U.S. Food and Drug Administration (FDA) and then approved by the European drug administration (EMA), is a humanized monoclonal antibody, targeted to calcitonin gene-related peptide (CGRP), approved for the treatment of migraine in adult patients (Martin, V., Samaan, K.H., Aurora, S.et al. adv Ther 37, 2034-. According to the FDA approved drug insert (Label) for the above three marketed antibodies in the united states, both eptnizuzumab and galanazumab were injected subcutaneously once a month and Fremanezumab could be injected subcutaneously once a month or once every 3 months.

However, in the face of the medical needs of patients for disease treatment, especially the needs of antibody drugs, there is still a need for anti-CGRP antagonist antibodies with lower immunogenicity, longer half-life and better pharmaceutical effects.

Disclosure of Invention

The present invention provides antibodies or antigen-binding fragments thereof having novel amino acid sequences, including monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, Fab ', F (ab') 2, Fv, scFv, or dsFv fragments, and the like.

The invention provides an antibody or antigen binding fragment thereof, comprising:

1)3 light chain complementarity determining regions (LCDR), wherein the amino acid sequence of LCDR1 is shown as SEQ ID NO. 1, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 3; and 3 heavy chain complementary determining regions (HCDRs), wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8, preferably SEQ ID NO. 6; or

2) A variant relative to the combination of CDR sequences in 1), wherein said variant has 1-10 amino acid substitutions in any one or more of the CDR regions.

Such variants are those in which the amino acid in any one or more of the LCDR or HCDR regions is altered, preferably by amino acid substitution, more preferably by conservative substitution; for example, the LCDR1, LCDR2, and/or LCDR3 region of the light chain variable region comprises at least one and no more than 10 amino acid changes in total; and/or the HCDR1, HCDR2, and/or HCDR3 region of the heavy chain variable region collectively comprises at least one and no more than 10 amino acid changes.

In one aspect of the invention, the antibody or antigen binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID No. 9, or an amino acid sequence having at least 80%, at least 85%, or at least 90% identity to the amino acid sequence set forth in SEQ ID No. 9; also comprises a heavy chain variable region, the amino acid sequence of which is shown in any one of SEQ ID NO 10, SEQ ID NO 11 or SEQ ID NO 12, or the amino acid sequence of which has at least 80%, at least 85% or at least 90% identity with SEQ ID NO 10, SEQ ID NO 11 or SEQ ID NO 12. Preferably, the antibody or antigen-binding fragment thereof comprises the following light and heavy chain variable region sequences:

1) comprising the amino acid sequence shown in SEQ ID NO 9 and/or comprising the amino acid sequence shown in SEQ ID NO 10, or

2) A light chain variable region comprising the amino acid sequence shown in SEQ ID NO 9 and/or a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO 11, or

3) A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO 9 and/or a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 12.

In one aspect, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region comprises a gamma-1, gamma-2, gamma-3, or gamma-4 human heavy chain constant region or a variant of the human heavy chain constant region, preferably having the sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG. In one aspect, the antibody further comprises a light chain constant region, wherein the light chain constant region comprises a lambda or kappa human light chain constant region, preferably having the sequence TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

In one aspect of the invention, the binding antigen of the above antibody or antigen binding fragment thereof is CGRP, preferably, the CGRP is human CGRP.

The invention also provides a nucleic acid encoding an antibody or antigen-binding fragment thereof comprising any of the above, wherein the antigen-binding portion of the antibody or antigen-binding fragment thereof is a CGRP, particularly a human CGRP.

In one aspect of the invention, the nucleic acid encodes an antibody or antigen-binding fragment thereof comprising the amino acid sequence:

1) comprises 3 light chain complementary determining regions (LCDR), wherein, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 1, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 3; and 3 heavy chain complementarity determining regions (HCDRs), wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8, preferably SEQ ID NO. 6; or

2) A variant relative to the combination of CDR sequences in 1), wherein said variant has 1-10 amino acid substitutions in any one or more of the CDR regions.

The present invention relates to nucleic acids which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80%, identity between the two sequences. The invention relates in particular to nucleic acids which hybridize under stringent conditions with the nucleic acids according to the invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Furthermore, the hybridizable nucleic acid encodes a polypeptide having the same biological function and activity as the mature polypeptide.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The present invention also provides vectors comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof that bind CGRP, preferably, the vectors are expression vectors, including, but not limited to, viral vectors such as adenoviral vectors, retroviral vectors, adeno-associated viral vectors, and the like; non-viral vectors such as plasmids, transposon vectors, etc., wherein the plasmid vector is preferably pCDNA3.4(Life Technology) vector. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The invention also provides a cell for expressing an antibody or antigen-binding fragment thereof that binds CGRP, comprising an expression vector encoding the antibody or antigen-binding fragment thereof that binds CGRP or a nucleic acid encoding the antibody or antigen-binding fragment thereof that binds CGRP, preferably a host cell comprising the expression vector described above. In one aspect of the invention, host cells expressing an antibody or antigen-binding fragment thereof that binds CGRP include, but are not limited to, mammalian cells, insect cells, plant cells, fungal cells, prokaryotic cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc. Preferably, the host cell for expressing the CGRP binding antibody or antigen binding fragment thereof is HEK 293.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the antibody encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant antibody in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

According to another aspect of the present invention there is also provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, nucleic acid, vector or cell that binds CGRP as described above, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvent, dispersant, additive, plasticizer and medicinal auxiliary material. Generally, these materials are non-toxic, inert and pharmaceutically acceptable carrier media. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or, e.g., topical administration (e.g., injection).

The invention also relates to a kit comprising any of the above-described antibodies or antibody fragments thereof, nucleic acids that bind to CGRP antigen. In one aspect of the invention, the kit comprises an antibody or antigen-binding fragment thereof comprising any one of the following sets of CDR amino acid sequences: LCDR1 shown in SEQ ID NO. 1, LCDR2 shown in SEQ ID NO. 2, LCDR3 shown in SEQ ID NO. 3, HCDR1 shown in SEQ ID NO. 4, HCDR2 shown in SEQ ID NO. 5, HCDR3 shown in SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO. 8. In one aspect of the invention, the kit further comprises a detection reagent for detecting the CGRP antigen-antibody reaction, a negative control and a positive control.

The antibody or the antigen binding fragment thereof has wide biological application value and clinical application value, and the application relates to the fields of diagnosis and treatment of CGRP related diseases, basic medical research, biological research, detection and the like. One preferred application is for clinical diagnosis and targeted therapy against CGRP.

In another aspect, the invention relates to the use of an antibody or antigen-binding fragment thereof, a nucleic acid, a vector or a cell according to any one of the preceding aspects in the preparation of a pharmaceutical composition for the treatment or prevention of a disease.

In another aspect, the invention relates to the use of the antibody or antigen binding fragment thereof, nucleic acid of any one of the preceding aspects in the preparation of a diagnostic or detection kit.

In another aspect, there is provided a method of treating or preventing a disease, comprising administering to a subject in need thereof an antibody or antigen-binding fragment, nucleic acid, vector, cell, or pharmaceutical composition of the invention.

In another aspect, a method of diagnosis, detection is provided, comprising administering an antibody or antigen-binding fragment, nucleic acid, or kit of the invention to a subject or sample in need thereof.

In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell or pharmaceutical composition of any of the preceding aspects for the treatment, prevention or treatment of a disease.

In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof, a nucleic acid, or a kit of any of the preceding aspects for detection, diagnosis.

In one aspect of the invention, the disease is preferably a CGRP related disease, further preferably the CGRP related disease is headache, more preferably migraine (miravine), including episodic migraine (i.e. 4-14 attacks per month) and chronic migraine (i.e. >14 attacks per month).

The inventor successfully obtains a kind of anti-CGRP antibody through extensive and intensive research and a large number of screens, and experimental results show that the CGRP antibody obtained by the invention can effectively block the interaction between the CGRP and the receptor thereof, and surprisingly, the CGRP antibody optimized by the invention can effectively block the combination between the CGRP and the receptor thereof, and the optimized CGRP antibody has high affinity, low immunogenicity, long in-vivo half-life and remarkable inhibition effect on vasodilatation. The present invention has been completed based on this finding.

Definitions and explanations

As used herein, the following terms and phrases are intended to have the following meanings, unless otherwise indicated. A particular term or phrase is not to be considered as indefinite or unclear unless it is specifically defined, but rather construed according to ordinary meaning.

The antibodies of the invention are designed with engineered CDRs and have portions (framework, hinge and constant regions, all or part) of antibodies of human origin that are identical or substantially identical (substantially human) to the framework and constant regions derived from human genomic sequences. The fully human framework, hinge region and constant regions are human germline sequences as well as sequences with naturally occurring somatic mutations and sequences with engineered mutations. The antibodies of the invention may comprise a framework, hinge or constant region derived from a fully human framework, hinge or constant region comprising one or more amino acid substitutions, deletions or additions therein. Furthermore, the antibodies of the invention are preferably substantially non-immunogenic in humans.

The antibodies of the invention are IgG-type antibodies and have "heavy" chains and "light" chains that are cross-linked via intra-and inter-chain disulfide bonds. Each heavy chain comprises an N-terminal HCVR (or VH) and a heavy chain constant region ("HCCR"). Each light chain comprises an LCVR (or VL) and a light chain constant region ("LCCR").

The HCVR and LCVR regions can be further subdivided into hypervariable regions (referred to as complementarity determining regions ("CDRs")) interspersed with more conserved regions (referred to as framework regions ("FRs")). Each HCVR and LCVR consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. Herein, the three CDRs of the heavy chain are referred to as "HCDR 1, HCDR2 and HCDR 3" and the three CDRs of the light chain are referred to as "LCDR 1, LCDR2 and LCDR 3". The CDRs contain most of the residues that form interactions with the antigen.

The terms "CGRP" and "CGRP α" in the present invention are synonymous without specific description, and refer to any CGRP molecule or mutant to which the antibody of the present invention can bind.

Unless otherwise indicated, all references to candidate antibodies or short references thereof herein have the same meaning and refer to the same candidate antibody having the same light chain variable region and heavy chain variable region, and are used interchangeably. Illustratively, candidate antibodies, as they are named collectively under the CGRPM5-BA51.23 or CGRP-BA51.23-IgG2, may be referred to in this patent using the abbreviation "BA 51.23".

The term "encoded nucleic acid" may be a nucleic acid that includes the encoded nucleic acid, and may also include additional coding and/or non-coding sequences.

The "fusion protein" of the antibody or antigen-binding fragment thereof of the present invention refers to a protein form formed by connecting the antibody or antigen-binding fragment thereof of the present invention to other proteins by any means conventional in the art, including but not limited to: adding protein genes to form a long open reading frame, thereby expressing the corresponding fusion protein in a gene expression mode; it is also possible to express proteins or protein units individually and then to link these proteins or building blocks together in a specific manner, for example by modifying the corresponding protein units to comprise partners with good covalent binding capacity, such as biotin/avidin (biotin/avidin), which makes it possible for the proteins to form fusion proteins by non-covalent interactions.

"conservative substitution" as used herein refers to the replacement of an amino acid in a protein or polypeptide structure by another amino acid that is physically or chemically similar to the amino acid, such that changes to the protein or polypeptide generally have little or no effect on the function of the protein. It is to be understood that any amino acid substitution having the above-mentioned characteristics is within the meaning of "conservative substitution" as described herein.

Drawings

FIG. 1 shows the serum titer of BALB/c mice after triple immunization, the serum titer being 12500-fold dilution.

FIG. 2 shows serum titers of Boan-hMab mice after seven immunizations, which were 2500-fold dilutions.

FIG. 3 shows the binding activity of Elisa assay candidate antibody to CGRP alpha protein

FIG. 4 shows the sensitivity of Elisa detection of binding of candidate antibodies to CGRP α

FIG. 5 shows the binding sensitivity of the optimized BA51.23 antibody to CGPP α

FIG. 6 shows the detection of the binding activity of the optimized BA51.23 antibody to human CGRP

FIGS. 7A-C are graphs showing the inhibition of capsaicin-stimulated vasodilation by the optimized BA51.23 antibody after 48h (FIG. 7A), 96h (FIG. 7B), 168h (FIG. 7C) of vehicle/antibody subcutaneous injection at a dose of 10mg/kg as a function of time

FIGS. 8A-C are AUC plots showing inhibition of capsaicin-stimulated vasodilation by optimized BA51.23 antibody following subcutaneous vehicle/antibody administration at a 10mg/kg dose for 48h (FIG. 8A), 96h (FIG. 8B), 168h (FIG. 8C)

FIG. 9 shows the optimized BA51.23 antibody in vivo potency curve (5mg/kg)

FIG. 10 shows the detection of the immunogenicity of CGRPM5-BA51.23-IgG2-N105D antibody

Detailed Description

The invention will be better understood with reference to the following examples. However, it is to be understood that the following examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way.

Example 1 Generation of anti-CGRP monoclonal antibodies

1.1 immunization

Wild type mouse immunization: (ii) human CGRP polypeptide (GenScript Synthesis) (II)https://www.ncbi.nlm.nih.gov/prote in/1005250Areport＝genbank&log$＝protalign&blast_rank＝14&RID＝907JCDCA014#feature_100 5250A(ii) a NCBI access: 1005250A) was coupled to KLH protein and then immunized against BALB/c mice after emulsification with Freund's adjuvant. The first immunization uses Freund's complete adjuvant, and the second to third immunization uses Freund's incomplete adjuvant, and 5 mice are immunized in total. Mice with higher serum titers were selected for boosting, and the spleens were removed from the sacrificed mice after 4 days for subsequent experiments. Serum titers after immunization of mice are shown in figure 1.

Immunization of transgenic mice: human CGRP polypeptide (synthesized by GenScript) is coupled to KLH protein, and is emulsified with gold adjuvant to immunize Boan biological full human antibody transgenic mouse Boan-hMab. This time, 10 mice were immunized in total, and this time, eight times. Serum titers after immunization of mice are shown in figure 2.

1.2 phage library creation

Taking spleen cells of an immune mouse, adding Trizol (Thermo Scientific, catalog number 15596-026), adding 1/5 volumes of chloroform after full lysis, fully mixing, standing at room temperature for 20min, centrifuging at 4 ℃ 12000rpm for 20min, taking an upper layer aqueous solution, adding isopropanol with the same volume, standing at room temperature for 20min, centrifuging at 4 ℃ 12000rpm for 20min, discarding a supernatant aqueous solution, adding 75% ethanol for washing twice, centrifuging at 4 ℃ 12000rpm for 5min, discarding an aqueous solution, keeping a precipitate, air-drying at room temperature, adding DEPC water for re-suspending the precipitate to obtain RNA, and carrying out reverse transcription on the RNA into cDNA by using a Roche Applied Science, catalog number 4897030001) according to the specification. Phage library establishment procedures reference Carlos f. barbas III, Phage display: the method described in A laboratory manual, obtaining the variable regions of the heavy chain and the light chain from cDNA by PCR method, obtaining ScFv by overlap extension PCR method from the variable regions of the heavy chain and the light chain, connecting the ScFv with plasmid pCOMB3x after enzyme digestion, then electrotransfecting the connection product into Escherichia coli TG1 competent cells, adding phage after TG1 is cultured for infection, and then recovering the culture supernatant. Phage library established with mouse accession number CGRPM05 CGRPM05, library volume 2.4X 10⁸(ii) a Phage library established with mouse accession number CGRPM08 CGRPM08, library volume 1.7X 10⁸。

1.3 screening was carried out in two ways

2 methods are used for flat screen, the first method is as follows: CGRP (1-37) protein (synthesized by GenScript) coated onto plates at 1. mu.g/well, left overnight at 4 ℃, plates were blocked with 3% skim milk for 1h the following day, and phage library (2X 10) was added¹²) Incubating for 2h, washing for 4-10 times, and eluting CGRP-bound phage by using an Elution Buffer (pH 2.2); the second method comprises the following steps: neutroavidin protein (Thermo, 31000) at 1. mu.g/plate, left overnight at 4 ℃ and then administeredBlocking the plate with 3% skimmed milk for 1h, adding 3. mu.g/library of Biotin-CGRP (1-37) (GenScript Synthesis), incubating for 1h, and adding phage library (2X 10)¹²) Incubation for 2h, washing 4-10 times followed by Elution of CGRP-bound phage using an Elution Buffer (pH 2.2).

And (2) magnetic bead screening, namely, combining the Biotin-CGRP (1-37) with magnetic beads (Invitrogen Dynabeads M-280 Streptavidin, 00355871) for 1h, then incubating with a phage library, washing for 4-10 times, and eluting with an resolution Buffer (pH 2.2) to obtain CGRP combined clones.

The magnetic bead screening obtains clones CGRPM5-BA51-IgG2 and CGRPM1-BA219-IgG2 (hereinafter abbreviated as BA51 and BA219), wherein CGRPM1 represents the immunized 1 st wild-type mouse, and BA represents the magnetic bead screening.

1.4 hybridoma selection

Dissecting CGRPQ40/Q42 mouse, collecting spleen to obtain B cell suspension, collecting 1x10 each of CGRPQ40/Q42 spleen cells⁷cells and 1x10⁷The SP2/0 cells are electrofused, the semi-solid is spread on a dish on the next day, after 10 days, a single clone is picked to a 96-well plate, and the diluted positive cells are added into the 96-well plate by using a limiting dilution method, wherein the four gradients are as follows: 8cells/well, 4cells/well, 2cells/well, 1 cell/well, cultured at 37 ℃ for 9 days with 5% CO 2; 8 subclones are selected from each clone and are placed in 96w, the positive subclones after cell activity detection are transferred into a 6-well plate, the subcloned RNA is extracted, cDNA is obtained after reverse transcription, and the heavy chain and light chain variable regions of each subclone are obtained by a PCR method and are used for constructing and producing antibody molecules. CGRPQS 89-2.3-antibody (QS 89-2.3) is obtained by screening in the method.

1.5 construction and production of antibody molecules

Clones BA51, BA219, QS89-2.3 were sequenced to Invitrogen Biotechnology Ltd and the amino acid sequences of the antibodies are shown in Table 1.

TABLE 1 active cloning of amino acid sequences

The antibody variable region gene was amplified by conventional molecular biology PCR (2. Phanta Max Master Mix manufacturer: Vazyme cat # P515-AA lot # TE211G8), the signal peptide was ligated to the variable region gene by overlap extension PCR, the antibody heavy chain variable region gene was ligated to vector pCDNA3.4(Life Technology) carrying antibody Fc (IgG2 subtype) sequence, respectively, by homologous recombination (Cloneexpress II One Step Cloning Kit manufacturer: Vazyme cat # C112-01 lot # TE211L8), and the antibody light chain variable region gene was ligated to vector pCDNA3.4 carrying antibody light chain constant region sequence, as shown below.

Light chain constant region sequence:

heavy chain constant region sequence:

after extracting plasmids from sequenced positive clones, cotransfecting the positive clones into HEK293 cells, culturing the cells in a shaker at 37 ℃ and 8% CO 2/125 rpm, performing Protein A affinity chromatography on the supernatant after 7 days of transient expression, purifying to obtain CGRP antibodies, and determining the antibody concentration by UV280 combined with theoretical extinction coefficient.

Control antibody production: the amino acid sequence of the CGRP antibody Fremanezumab was determined by IMGT database and patent CN 200680042443. The whole gene is synthesized and inserted into the carrier pCDNA3.4 to be expressed by HEK293 cells, and the produced antibody is named CGRP-TEVA-IgG 2.

The amino acid sequence of the CGRP antibody, eptiuzumab, was determined by IMGT database and patent CN 201280035852. The whole gene is synthesized and inserted into the carrier pCDNA3.4 to be expressed by HEK293 cells, and the produced antibody is named CGRP-Alder-IgG 2.

The amino acid sequence of the CGRP antibody galcanezumab was determined by IMGT database and patent CN 201180028611. The whole gene is synthesized and inserted into a vector pCDNA3.4 to be expressed by HEK293 cells, and the produced antibody is named CGRP-Lilly-IgG 2.

TABLE 2 control antibody amino acid sequences

1.6 detection of the binding Activity of antibodies to CGRP alpha Using Elisa

In order to determine the binding capacity of the obtained antibody and antigen, the binding condition of the antibody and CGRP alpha with different concentrations is detected by Elisa, so that the antibody with stronger binding capacity with the CGRP alpha is obtained.

Coating the antigen CGRP alpha (4013281-1000; Bachem) with different concentrations (0.2 mu g/mL, 0.1 mu g/mL, 0.05 mu g/mL, 0.025 mu g/mL, 0.0125 mu g/mL, 0.00625 mu g/mL, 0.003125 mu g/mL, 0 mu g/mL), 100 mu L/hole for 4 degrees overnight; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100ul of 1. mu.g/mL candidate antibody (antibody stored in 25mM Tris, 0.1M Gly, 20mM NaCl, pH6.5 buffer) per well, and incubating at 37 deg.C for 1 h; then adding goat anti-human IgG/HRP, incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on a microplate reader.

The results are shown in FIG. 3, and the antibodies CGRPM5-BA51-IgG2, CGRPM1-BA219-IgG2 and CGRP QS89-2.3-IgG2 all have higher binding ability with antigen in the concentration range shown in the experiment.

Example 2 obtaining of humanized antibody

2.1 antibody humanization

The three-dimensional structure simulation of the antibody is carried out by using Discovery Studio software, the humanized transformation is carried out on the murine sequence by combining CDR graft, the back mutation sites are determined by back mutation analysis, and finally the antibodies CGRPM5-BA51.23, BA51.27, CGRPM1-BA219.53 and BA219.54 (hereinafter referred to as BA51.23, BA51.27, BA219.53 and BA219.54 in the invention) are obtained. The BA51.23 clone was sequenced to determine that the heavy chain was of the germline gene IGHV1-2 family and the light chain was of the germline gene IGKV1-33 family. The sequence is shown in Table 3.

TABLE 3 humanized antibody amino acid sequences

The antibody sequence is inserted into a corresponding vector through conventional molecular biology techniques such as amplification and the like, cells are transfected and cultured, and the obtained antibody is purified and detected, and the specific steps refer to the experimental contents in section 1.5 of the invention.

Example 3 characterization of candidate antibodies

3.1 Elisa detection of binding of candidate antibodies to CGRP alpha protein

The binding capacity of the candidate antibody and CGRP alpha with different concentrations is detected by Elisa, and an antibody with stronger binding capacity with the CGRP alpha is expected to be obtained.

Coating the antigen CGRP alpha (4013281-1000; Bachem) with different concentrations (0.2 mu g/mL, 0.1 mu g/mL, 0.05 mu g/mL, 0.025 mu g/mL, 0.0125 mu g/mL, 0.00625 mu g/mL, 0.003125 mu g/mL, 0 mu g/mL), 100 mu L/hole for 4 degrees overnight; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; mu.l each of 1. mu.g/ml candidate antibody (antibody stored in 25mM Tris, 0.1M Gly, 20mM NaCl, pH6.5 buffer) was added to each well and incubated at 37 ℃ for 1 h; then adding goat anti-human IgG/HRP, incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on a microplate reader. The results are shown in FIG. 4 and Table 4.

The results show that CGRPM5-BA51.23-IgG2, CGRPM5-BA51.27-IgG2 have close sensitivity to the control antibodies CGRP-TEVA-IgG2, CGRP-LILY-IgG 2.

TABLE 4 Elisa detection sensitivity of candidate antibodies in binding to CGRP alpha

Sample (I)	EC50(μg/mL)
CGRPM5-BA51.23-IgG2	0.036
CGRPM5-BA51.27-IgG2	0.034
CGRP-QS89-2.3-IgG2	0.083
CGRP-TEVA-IgG2	0.040
CGRP-ALD-IgG2	0.031
CGRP-LILY-IgG2	0.037

3.2 BLI detection of binding of candidate antibodies to CGRP proteins

Antibody binding kinetics Using biomembrane interference-based technique (Biolayer interference BLI) Octet^REDAnd measuring by using a 96 instrument. Coupling of alpha-CGRP (0.1. mu.g/mL) to Streptavidin (SA) Dip and Read^TMOn Biosensors, loading height 0.19nm, 2-fold serial dilutions of antibody (antibody stored in 25mM Tris, 0.1M Gly, 20mM NaCl, pH6.5 buffer) in PBST, 50mM start and set 0 concentration, Association time set at 300s, and Association time set at 900 s. After completion of the detection, the binding constant (kon) and dissociation constant (kdis) were calculated using Curve Fitting of 1:1Model, and the equilibrium dissociation constant (kD) was calculated as the ratio kD/ka. The results are shown in Table 5.

The results show that: the antibodies CGRPM5-BA51.23-IgG2 and CGRPM5-BA51.27-IgG2 have weaker dissociation tendency compared with CGRP-TEVA-IgG2, and the affinity of CGRPM5-BA51.23-IgG2 and CGRPM5-BA51.27-IgG2 is stronger than that of CGRP-TEVA-IgG 2.

TABLE 5 Octet detection of candidate antibody binding kinetics

Antibody ID	Kon(1/Ms)	Kdis(1/s)	KD(M)
CGRPM5-BA51.23-IgG2	1.22E+05	<1.0E-07	<1.0E-12
CGRPM5-BA51.27-IgG2	1.62E+05	<1.0E-07	<1.0E-12
CGRP-TEVA-IgG2	5.93E+05	1.60E-05	2.71E-11
CGRP-QS89-2.3-IgG2	2.02E+05	6.42E-05	3.18E-10

Example 4 BA51.23 antibody optimization

BA51.23 antibody sequence optimization

In order to further obtain CGRP antibody molecules with better effect, the molecular biology technology is adopted to carry out the following stepsHeavy chain N of CGRPM5-BA51.23-IgG2¹⁰⁵Respectively mutated to D¹⁰⁵And L¹⁰⁵. CGRPM5-BA51.23-IgG2-N105D light chain variable region sequence is shown in SEQ ID NO:9 (LCDR1-3 is shown in SEQ ID NO: 1-3) and heavy chain variable region is shown in SEQ ID NO:10 (HCDR1-3 is shown in SEQ ID NO: 4-6); CGRPM5-BA51.23-IgG2-N105L light chain variable region sequence is shown in SEQ ID NO:9 (LCDR1-3 is shown in SEQ ID NO: 1-3) and heavy chain variable region is shown in SEQ ID NO:11 (HCDR1-3 is shown in SEQ ID NO: 4/5/7). The antibody sequence is inserted into a corresponding vector through conventional molecular biology techniques such as amplification and the like, cells are transfected and cultured, and the obtained antibody is purified and detected, and the specific steps refer to the experimental contents in section 1.5 of the invention.

4.2 detection of optimized antibody binding sensitivity to CGRP alpha Using Elisa

The binding capacity of the optimized antibody and the CGRP alpha with different concentrations is detected by Elisa, and the antibody with stronger binding capacity with the CGRP alpha is expected to be obtained.

Coating the antigen CGRP alpha (4013281-1000; Bachem) with different concentrations (0.2 mu g/mL, 0.1 mu g/mL, 0.05 mu g/mL, 0.025 mu g/mL, 0.0125 mu g/mL, 0.00625 mu g/mL, 0.003125 mu g/mL, 0 mu g/mL), 100 mu L/hole for 4 degrees overnight; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; add 1. mu.g/ml candidate antibody (samples in 25mM Tris, 0.1M Gly, 20mM NaCl, pH6.5 buffer) 100. mu.L each per well, incubate 1h at 37 ℃; then adding goat anti-human IgG/HRP, incubating for 1h at 37 ℃, developing for 10min, and reading OD450 on a microplate reader. The results are shown in FIG. 5 and Table 6:

the results show that the optimized antibodies CGRPM5-BA51.23-IgG2-N105D and CGRPM5-BA51.23-IgG2-N105L have stronger binding capacity than CGRPM5-BA51.23-IgG 2.

TABLE 6 binding sensitivity EC50 of BA51.23 to CGPP α after optimization

ID	EC50(ng/ml)
CGRPM5-BA51.23-IgG2-N105L	10.59
CGRPM5-BA51.23-IgG2-N105D	8.437
CGRPM5-BA51.23-IgG2	13.45

4.3 detection of the binding Activity of the optimized BA51.23 antibody and CGRP

The in vitro function of CGRP antibodies was tested using cell-based cAMP activation and binding assays. Cells were diluted with Stimulation Buffer, 4X 10⁵Cells/mL, added to a white 384-well plate at 5. mu.L/well, 2000/well. Samples (samples stored in 25mM Tris, 0.1M Gly, 20mM NaCl, pH6.5 Buffer) were diluted to 200. mu.g/mL using a Stimulation Buffer, and 2-fold dilutions were performed sequentially for a total of 8 concentration gradients and added to the wells at 2.5. mu.L/well. CGRP was diluted to 2. mu.g/mL with Ststimulation Buffer and added to the cell wells at 2.5. mu.L/well. Incubate at room temperature for 1.5 h. cAMP-D2 was diluted 5-fold with lysine buffer and added to the cell wells at 5. mu.L/well. Anti-cAMP-Cryptate was diluted 5-fold with lysine buffer and added to the wells at 5. mu.l/well. Incubate for 1h at room temperature in the dark. Reading with a microplate reader HTRF module. The results are shown in FIG. 6 and Table 7:

the results of the determination of cell functional activity show that the optimized antibodies have good CGRP binding activity.

TABLE 7 detection of binding Activity of BA51.23 and CGRP after optimization EC50

ID	EC50(μg/mL)
CGRPM5-BA51.23-IgG2-N105L	44.85
CGRPM5-BA51.23-IgG2-N105D	32.00

4.4 pharmacodynamic study of optimized BA51.23 antibody in SD rat capsaicin-stimulated vasodilation model

The functional study of the CGRP antibody was performed using the SD rat capsaicin-stimulated vasodilation model. SD rats (6 rats per group) were deeply anesthetized by intraperitoneal injection of pentobarbital sodium (50-60mg/kg) and placed in a constant temperature blanket. The vehicle/antibody (vehicle component: 10mM CH) was administered by subcutaneous injection at a dose of 10mg/kg₃COONa■3H ₂O, 30mM NaCl, 0.03% Tween-20, 5% sucrose, ph6.0, antibody stored in vehicle) for 48h, 96h, 168h, a baseline level measurement of dorsal skin blood flow was performed, and blood flow was continued after subcutaneous capsaicin injection. The laser Doppler skin blood flow measuring probe is firmly adhered to the skin to be measured by using a double-sided adhesive tape for blood flow measurement, a laser Doppler blood flow meter and recording software are opened, a stable blood flow baseline is recorded for 10min, capsaicin is injected subcutaneously into the skin to be measured, measurement is continued for 20-30 min, the average blood flow value per minute is taken as a Perfusion Unit (PU), and the result is shown in figures 7A-7C. The area under the curve, AUC, was obtained by time x rate of change in blood flow, reflecting the effect of blood flow inhibition, and the results are shown in FIGS. 7A-7C and FIGS. 8A-8C.

According to the information of the FDA published drug instruction, the ratio of the reduction of the monthly migraine days of the CGRP-Alder-IgG2 by more than 50 percent can reach more than 60 percent under the dosage of 300mg, and the in vivo efficacy is better than that of other control antibodies, so the CGRP-Alder-IgG2 is selected as the control antibody of the experiment. The results show that the AUC of the blood flow assay of CGRP-Alder-IgG2 after 96h administration is 1.36 times that of CGRPM5-BA51.23-IgG 2-N105D; the AUC of the blood flow assay of CGRP-Alder-IgG2 after 168 hours of administration was 1.82 times that of CGRPM5-BA51.23-IgG 2-N105D; the demonstration shows that CGRPM5-BA51.23-IgG2-N105D has more obvious inhibition effect on capsaicin-stimulated vasodilatation compared with other antibodies such as CGRP-Alder-IgG2 and the like, and indicates that CGRPM5-BA51.23-IgG2-N105D has better clinical treatment effect.

4.5 study of CGRP antibodies on cynomolgus monkey PK

Cynomolgus monkeys (2 per group) were given subcutaneous injections of different CGRP antibodies (antibody stock in 10mM CH) at a dosing dose of 5mg/kg₃COONa■3H ₂O, 30mM NaCl, 0.03% Tween-20, 5% sucrose, pH 6.0), and before (0h) and after (1 h, 4h, 10h, 1d, 2d, 3d, 4d, 5d, 7d, 10d, 14d, 21d and 28d of administration, taking out whole blood samples through veins, placing the whole blood samples in a blood sample collection tube, naturally coagulating the whole blood samples in an ice box, placing the whole blood samples in a centrifuge for 8h after blood sample taking, centrifuging for 10min at 1000-3000 g, separating serum, placing the serum in a sample preservation tube, and detecting the metabolism condition of the antibody in the cynomolgus monkey by using the method of Elisa. The results are shown in FIG. 9 and Table 8.

The dosing interval of CGRP-TEVA-IgG2 was up to three months, so we chose CGRP-TEVA-IgG2 as the control antibody for this experiment. The result shows that compared with CGRP-TEVA-IgG2, CGRPM5-BA51.23-IgG2-N105D has longer half-life, which indicates that CGRPM5-BA51.23-IgG2-N105D has more durable therapeutic effect, and CGRPM5-BA51.23-IgG2-N105D can prolong the administration period, reduce the treatment cost and greatly relieve the economic pressure of patients on the basis of once administration for three months of Fremanezumab; meanwhile, compared with CGRP-TEVA-IgG2, CGRPM5-BA51.23-IgG2-N105D can improve AUC and has higher bioavailability.

TABLE 8 metabolism of candidate antibodies in cynomolgus monkeys

Parameter	Unit	CGRP-TEVA-IgG2	CGRPM5-BA51.23-IgG2-N105D
t 1/2	h	138.6	195.7
C max	μg/mL	56.1	52.6
T max	h	36.0	36.0
AUC (0-t)	μg/mL*h	11460.8	13081.0
AUC (0-∞)	μg/mL*h	12017.3	15001.7
V z/F	mL/kg	81.6	95.0
CL z/F	mL/h/kg	0.42	0.388
MRT (0-t)	h	153.1	204.0

4.6 detection of immunogenicity of CGRP antibodies

The content of the anti-drug antibody in monkey sera (monkey sera from example 4.5)) at different time points after subcutaneous administration of 5mg/kg antibody was determined by the method of Elisa. Coating CGRP-TEVA with CBS coating solution (pH9.6 carbonic acid solution) at 0.125 μ g/mL, CGRPM5-BA51.23-IgG2-N105D 0.0625 at 0.0625 μ g/mL, 100 μ L/well 4 degree overnight; sealing with 3% skimmed milk powder at 37 deg.C for 1 hr; adding 100X cynomolgus monkey serum into each hole, incubating for 1h at 37 ℃ with 100 mu L; adding CGRP-TEVA-biotin 0.125 μ g/mL, CGRPM5-BA51.23-IgG2-N105D-biotin 0.0625 μ g/mL, incubating at 37 ℃ for 1h, washing, adding streptomycin/HRP, and incubating at 37 ℃ for 1 h; after TMB development for 10min, OD450 was read on the microplate reader. The results are shown in FIG. 10.

The results showed that neither CGRPM5-BA51.23-IgG2-N105D nor CGRP-TEVA-IgG2 detected immunogenicity.

Claims (10)

1. An antibody or antigen-binding fragment thereof, comprising:

   1)3 light chain complementarity determining regions, wherein the amino acid sequence of LCDR1 is shown as SEQ ID NO. 1, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 3; and

   3 heavy chain complementarity determining regions, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8, and preferably SEQ ID NO. 6; or

   2) A variant relative to the combination of CDR sequences in 1), wherein said variant has 1-10 amino acid substitutions in any one or more of the CDR regions.
2. An antibody or antigen-binding fragment thereof, comprising a light chain variable region having an amino acid sequence with at least 80%, at least 85%, or at least 90% identity to the amino acid sequence set forth in SEQ ID No. 9 and/or a heavy chain variable region comprising an amino acid sequence with at least 80%, at least 85%, or at least 90% identity to the amino acid sequence set forth in any one of SEQ ID No. 10, SEQ ID No. 11, or SEQ ID No. 12.
3. The antibody or antigen-binding fragment thereof of claim 2, comprising a light chain variable region having the amino acid sequence of SEQ ID No. 9; and a heavy chain variable region having an amino acid sequence of SEQ ID NO 10 or SEQ ID NO 11 or SEQ ID NO 12, preferably the heavy chain variable region has an amino acid sequence of SEQ ID NO 10.
4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof binds antigen is CGRP.
5. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4.
6. A vector comprising the nucleic acid of claim 5; preferably, the vector is an expression vector.
7. A cell comprising the nucleic acid of claim 5 or the vector of claim 6.
8. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the nucleic acid of claim 5, or the vector of claim 6, or the cell of claim 7, and a pharmaceutically acceptable carrier.
9. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the nucleic acid of claim 5.
10. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or a nucleic acid according to claim 5, for the prevention, treatment, detection or diagnosis of a disease associated with CGRP, preferably wherein the CGRP-associated disease is headache, more preferably migraine.

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